Clinical Hematology

Hematology refers to the study of the numbers and morphology of the cellular elements of the blood—the red cells (erythrocytes), white cells (leukocytes), and platelets (thrombocytes) and the use of these results in the diagnosis and monitoring of disease. (Also see Hematopoietic System Introduction.)

Red Blood Cells

Three RBC measurements are routinely carried out: packed cell volume (PCV), the proportion of whole blood volume occupied by RBC; hemoglobin (Hgb) concentration of whole lysed blood; and RBC count, the number of RBC per unit volume of whole blood. Although these are separate estimations, they are in effect 3 ways of measuring the same thing, and it is incorrect to attempt to interpret them as separate variables. Inasmuch as they do vary in relation to each other, they allow calculation of 2 further meaningful parameters, mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC). See equations, below.

MCV varies widely between mammalian species, from ∼15 fL in goats to ∼90 fL in humans. Avian and reptilian red cells are even larger, up to 300 fL. Nevertheless, MCHC varies little with species (or erythrocyte size), at ∼330 g/L.

Several artifacts can cause significant and potentially misleading alterations to measured erythrocyte parameters: 1) old samples cause RBC to swell, thus increasing PCV and MCV and decreasing MCHC; 2) lipemia causes a falsely high Hgb reading, and hence a falsely high MCHC; 3) hemolysis causes PCV to decrease while Hgb remains unchanged, again leading to a falsely high MCHC; 4) underfilling of the tube causes RBC to shrink, causing PCV and MCV to decrease and MCHC to increase; 5) auto-agglutination causes a falsely low RBC count, and hence a falsely high MCV.

Visual description of RBC morphology on a Romanowsky stain also provides useful diagnostic information. The most common terms employed include: 1) Normocytic—cells are of normal size. 2) Macrocyte—abnormally large cell, usually polychromatophilic (see below). 3) Microcyte—abnormally small cell, usually caused by a lack of hemoglobin precursors. 4) Anisocytosis—variation in size of cells, due to macrocytes, microcytes, or both. 5) Normochromic—cells are of normal color. 6) Polychromasia—variation in color of the cells. This usually describes the appearance of large, juvenile, bluish-staining polychromatophilic macrocytes. These broadly correspond to the “reticulocyte” seen with new methylene blue staining, in which the reticulum represents the remnants of the nucleus. 7) Hypochromasia—decrease in staining density of the cells, usually due to a lack of hemoglobin precursors, especially iron. 8) Annulocyte—extreme form of hypochromic cell with only a thin rim of hemoglobin.

PCV is the variable normally used to assess the basic status of the erythron—increased in polycythemia, decreased in anemia—although if a sample is too hemolyzed to allow measurement of PCV, a meaningful Hgb measurement may still be obtained. RBC count as such should not be interpreted clinically.

An abnormally high PCV (polycythemia) may be relative, due to a change in the proportion of circulating RBC to blood plasma without any alteration in the size of the erythron, or absolute, due to a real increase in erythron size. Absolute polycythemia may be primary (eg, polycythemia vera or, rarely, erythropoietin-producing tumors) or secondary (a consequence of disease in another organ system). (Also see Erythrocytosis and Polycythemia.)

Polycythemia vera and erythropoietin-producing tumors should only be suspected when PCV is very high, normally >0.70. The former is characterized by normal adult erythrocytes and a normal (or low) erythropoietin concentration, while the latter may show a regenerative RBC picture with high erythropoietin concentration. Relative polycythemia may also be associated with very high PCV values, and normal adult RBC. Secondary polycythemia generally shows a more modest increase in PCV, often with evidence of regeneration (more so when the cause is pulmonary or cardiac, less so when the cause is hormonal). It is often possible to make the differential diagnosis of polycythemia on clinical grounds.

Abnormally low PCV (anemia) may be caused by loss of blood (hemorrhage), break-down of erythrocytes in circulation (hemolysis), or lack of production of erythrocytes by the bone marrow (hypoplasia or aplasia). Presentation varies according to whether the condition is acute or chronic. Aplastic anemia is always chronic in onset because anemia occurs gradually as existing cells reach the end of their lifespan. (Also see Anemia.)

In acute hemorrhagic anemia, external blood loss is easily appreciated clinically, but blood loss into a body cavity may only be determined on paracentesis. Initially, all hematologic parameters may be normal, as it may take 12 hr for fluid shifts to produce a decrease in the PCV. Within a few days, RBC become regenerative, with juvenile forms appearing in circulation (except in horses, where circulating evidence of regeneration is not readily appreciable). These consist of polychromatophilic macrocytes and normoblasts (nucleated RBC). Late normoblasts have a small, nonviable nucleus and a moderate amount of cytoplasm colored similarly to that of the polychromatophilic macrocytes, while early normoblasts have a larger, viable nucleus and scanty cytoplasm. These are most easily distinguished from lymphocytes by their more densely-staining nucleus.

If substantial amounts of blood have been lost from the body, the RBC picture may become hypochromic. Thus, this type of anemia shows an increase in MCV and a decrease in MCHC. If bleeding is into a body cavity, hypochromasia may not be evident because hemoglobin precursors will be recycled. However, slight jaundice may be seen as the sequestered cells are broken down. Some sequestered cells may also be returned to the circulation intact, if somewhat misshapen.

In acute hemolytic anemia, PCV will decrease immediately, and in the early stages some jaundice will be evident. In the very early stages, even a sample collected with extreme care may be markedly hemolyzed. As with hemorrhagic anemia, the RBC will become regenerative within a few days, with polychromatophilic macrocytes and nucleated RBC evident. Because hemoglobin precursors are not lost from the body, true hypochromasia is not seen.

Chronic hemorrhagic anemia may be difficult to appreciate if blood is lost in the feces or urine, or due to bloodsucking ectoparasites. Anemia may be severe, and the RBC picture will be regenerative on presentation. Hypochromasia is usually very marked. In very longstanding conditions, depletion of iron and other hemoglobin precursors can become so marked that most of the cells are microcytic, and MCV may paradoxically decrease. Intermittent intra-abdominal hemorrhage leads to a somewhat different picture, as blood shed into the peritoneal cavity can be returned to the circulation. PCV may therefore recover quickly, until the next episode, and signs of depletion of hemoglobin constituents do not emerge.

In chronic hemolytic anemia, RBC are regenerative on presentation, except that some cases of autoimmune hemolytic anemia (AIHA) paradoxically show little or no regeneration until treatment has been initiated. Hypochromasia is less marked than in hemorrhagic conditions, and misshapen erythrocytes (including target cells and folded cells) are more common. The spherocyte, in which the erythrocyte loses its classic biconcave shape, is essentially pathognomonic for AIHA. Jaundice may be absent, because the products of the destruction of the erythrocytes may be cleared by the reticuloendothelial system and the liver as quickly as they are formed.

Hypoplastic and aplastic anemia may be mild if RBC production is merely depressed secondary to some other disease. Protein, mineral, or vitamin deficiencies may cause hypoplastic anemia, but these are more likely to be secondary to another disease (eg, chronic hemorrhage or malabsorption) than simple dietary deficiency. Other diseases may cause depression of erythropoietin production, eg, renal failure, deficiencies of hormones that usually stimulate erythropoietin production (eg, hypothyroidism, Addison's disease), and chronic, debilitating conditions such as chronic infections, chronic parasitism, and neoplasia. RBC morphology is nonregenerative and may be hypochromic if a deficiency state is involved. Paradoxically, vitamin B12 and/or folic acid deficiency produces a macrocytic RBC picture due to early maturation arrest of the erythrocytes. Neoplasia of the bone marrow may cause severe anemia as erythropoietic elements are crowded out, but some regeneration may be observed as the remaining bone marrow attempts to compensate. In this case, other bone marrow cell lines will also be affected.

True aplastic anemia refers to a failure of the entire bone marrow. The shorter-lived granulocytes and platelets decrease first, followed by a progressively severe anemia that is normocytic and normochromic.

White Blood Cells

The white blood cells consist of the granulocytes (neutrophils in most mammals, heterophils in rabbits, reptiles, and birds; eosinophils; and basophils) and the agranulocytes (lymphocytes and monocytes). Although they are traditionally counted by determining each as a percentage of the total WBC population, meaningful interpretation requires that the absolute number of each type be calculated by multiplying the total white cell count by the fraction attributable to the individual cell type. An increased percentage that is due to an absolute decrease in another cell type is not an increase at all.

Mature neutrophils have a lobulated nucleus, but when demand is high immature cells with an unlobulated band nucleus may be released into circulation. They function as phagocytes and are important in infectious conditions and in inflammation. Increased neutrophil counts (neutrophilia) are caused by inflammation, bacterial infection, acute stress, steroid effects, and neoplasia of the granulocytic cell line (granulocytic leukemia can be difficult to differentiate from a simple neutrophilia without special stains or bone marrow biopsy). Decreased neutrophil counts (neutropenia) are caused by viral infections, toxin exposure (including foodborne toxins), certain drugs (eg, carbimazole and methimazole), autoimmune destruction of neutrophils, bone marrow neoplasia not involving the granulocytes, and bone marrow aplasia.

Eosinophils are characterized by prominent pink-staining granules on a Romanowsky stain. They inactivate histamine and inhibit edema formation. Increased eosinophil counts (eosinophilia) are caused by allergic/hypersensitivity reactions, parasitism, tissue injury, mast cell tumors, estrus, and pregnancy or parturition in the bitch. Some large continental dog breeds (eg, German and Belgian Shepherds, Rottweilers) normally have a relatively high eosinophil count. Extremely high eosinophil counts (hypereosinophilic syndrome), possibly due to an out-of-control hypersensitivity reaction, and eosinophil leukemia (a form of chronic myeloid leukemia) are also described. Decreased eosinophil count (eosinopenia) is almost always caused by the action of glucocorticoids, either endogenous or therapeutic.

Basophils are rare in most species and are characterized by blue-staining granules on a Romanowsky stain. They are closely related to mast cells and, like them, initiate the inflammatory response by releasing histamine. An increased basophil count (basophilia) accompanies eosinophilia in some species as part of the hypersensitivity reaction.

Monocytes are large cells with blue-gray cytoplasm, which may be vacuolated, and a kidney bean-shaped or lobulated nucleus. Their main function is phagocytosis, and they are essentially identical to tissue macrophages. An increased monocyte count (monocytosis) may occur in any chronic disease, especially chronic inflammation, and may be very marked in neoplasia. Monocytes also increase as part of the steroid response in dogs.

Lymphocytes mainly develop outside the bone marrow in the lymph nodes, spleen, and gut-associated lymphoid tissue. They are the smallest of the white cells, with a round, evenly staining nucleus and sparse cytoplasm. Their primary function is immunologic, including both antibody production and cell-mediated immune responses. Some survive only a few days, but many are long-lived. The number in circulation is a balance between populations in the blood, lymph, lymph nodes, and splenic follicles and does not necessarily reflect changes in lymphopoiesis. An increased lymphocyte count (lymphocytosis) may occur for physiologic reasons, especially in cats, but significant increases usually indicate leukemia. Immature or bizarre cells may also be recognized. Decreased lymphocyte counts (lymphopenia) are usually due to an effect of corticosteroids, either endogenous (stress or Cushing's disease) or therapeutic, and may also accompany neutropenia in some viral infections, especially the parvoviruses. Lymphopenia may also be a feature of solid-organ lymphosarcomas, when leukemia is absent.

Platelets

Mammalian platelets are pale blue granular fragments (much smaller than RBC) that are shed from multinucleate megakaryocytes in the bone marrow; avian and reptilian platelets are true cells with nuclei. They maintain the integrity of the endothelium and act as part of the clotting process to repair damaged endothelium, where they ensure mechanical strength of the clot.

Increased platelet counts (thrombocytosis) occur as a reaction to consumption following injury, when large juvenile platelets may also appear; after splenectomy, as splenic stores are liberated to the circulation; after vincristine treatment, which increases platelet shedding from megakaryocytes; and in megakaryocytic leukemia.

Decreased platelet counts (thrombocytopenia) are caused by autoimmune reactions, thrombotic/thrombocytopenic purpura, bone marrow suppression and aplasia, bone marrow neoplasia, and equine infectious anemia. Signs are petechiation and ecchymosis more than frank hemorrhage, and little may be seen until the platelet count is <20 × 109/L. Platelet functional abnormalities present similarly, but platelet numbers and morphology are normal.

Blood Sample Preparation and Evaluation

In-house hematologic investigations, with a minimum of specialist equipment, can provide almost as much information as a full laboratory analysis, although some estimations are qualitative rather than quantitative.

Blood for hematology should be collected into EDTA anticoagulant and immediately mixed well to avoid clotting. Larger (2.5 mL) tubes yield better results than the smaller (1 mL) pediatric tubes and are less likely to clot. Nevertheless, it is essential to fill the tube exactly to the mark, so smaller tubes may be unavoidable for small patients. White cell morphology deteriorates most quickly, especially in equine blood, so if the sample cannot be analyzed immediately, a thin blood smear should be submitted to the laboratory with the blood sample.

PCV is measured by microhematocrit, which is the reference method. A capillary tube is filled ¾ full with well-mixed blood and sealed at one end—heat-sealing is best if a bunsen burner is available, otherwise a proprietary clay pad is used. The tube is spun in a high-speed microhematocrit centrifuge for 6 min and the PCV is read using a microhematocrit reader with a sliding cursor. The appearance of the plasma (eg, normal, icteric, hemolyzed, lipemic) and the thickness of the buffy coat, which gives a very rough guide to total WBC count, should be noted.

Further information is obtained from a blood film. This is made by using one slide, with a corner broken off as a spreader, to pull a small drop of blood across a clean slide into a thin film. A suitable film is thin (1 erythrocyte thick) and tapers to a feathered edge before the far end of the slide. The broken corner of the spreader slide ensures 2 straight edges parallel to the long edges of the slide. Immediately after the film is spread, it is dried quickly by fanning the slide in the air. Air-dried smears can be sent to the laboratory in a slide mailer or stained and examined in the practice. Commercial rapid Romanowsky-type stains merely require the slide to be dipped in 3 colored solutions in succession. Cell morphology is clear and comparable to more permanent stains such as Leishman's or May-Gruenwald-Giemsa, although quality deteriorates after a few days. The slide should be allowed to dry naturally or dried with a hair-dryer (not wiped dry), and examined under oil immersion.

Clinically useful information may be easily achieved in-house for all hematologic variables. The main deficiency is the absence of a numerical WBC count, and hence of numerical values for the differential WBC count. This may be acceptable for an interim emergency investigation, and a WBC count using a hemocytometer slide may be attempted. A mirrored slide with an Improved Neubauer ruling may be used, with a cover slip fitted so that Newton's rings are visible on both sides. The blood sample is diluted 1:20 with Turck's fluid or a similar diluent. An automatic pipette capable of dispensing 0.95 mL and 0.05 mL (50 μL) should be used to ensure an accurate dilution. The sample should be mixed well and allowed to stand for 10 min while the stain is taken up by the cells. The chamber of the hemocytometer is then filled by using a capillary (PCV) tube. The number of cells in each of the 4 large corner squares of the grid are counted and the total divided by 20 to calculate the total WBC count × 10 9/L.

Several hematologic instruments are available for in-practice use. Those based on centrifugation, in which WBC measurements are made by spreading a stained buffy coat, are not true hematology analyzers, but give approximate counts. Although a numerical estimate of total WBC count is provided, the differential WBC count cannot distinguish between lymphocytes and monocytes, and results do not always correlate well with standard methods. This method is only appropriate as an emergency approximation, and a blood film must always be examined in addition. It is also wise to check the PCV by microhematocrit.

Impedance counters (Coulter principle) are used by professional laboratories and perform well in experienced hands. However, it is difficult to achieve optimal performance without trained technical staff. Non-Coulter-principle instruments should be avoided for veterinary use, as their results do not necessarily compare well on nonhuman samples. Instruments providing automated differential counts perform poorly on nonhuman blood, and results must never be accepted without also checking a blood film. No hematology laboratory should be without the facility for examining blood films, and a blood film should be examined for every patient sample. Quality assurance issues are the same as for biochemistry laboratories (see Clinical Biochemistry). If accuracy and reliability cannot be guaranteed to the same standard as the referral laboratory, then results should not be relied on without external confirmation.

Red Blood Cells

The size, uniformity of size, and presence of microcytes, macrocytes, and abnormally shaped cells should be noted, along with cell color, uniformity of color, and the presence of hypochromasia, polychromasia, and nucleated RBC. An overall descriptive assessment of the RBC picture should be made, including the degree of regeneration or hypochromasia, if any.

White Blood Cells

A qualitative estimate of numbers should be made (ie, very low, low, normal, raised, high, very high). This can be remarkably consistent with practice. The proportions of each cell type can be estimated, or preferably, a formal differential count of 100 or 200 cells performed. Unusual or abnormal white cell forms (eg, band or toxic neutrophils), or pathologic cells (eg, prolymphocytes, lymphoblasts, or mast cells) should be noted.

Platelets

A qualitative estimate of platelet numbers, based on how many can be seen in a typical high-power field (×1,000, oil immersion) should be made. Several fields should be examined, more if numbers appear low. Results can be ranked as none seen (on entire slide), rare (very few on entire slide), low in number (<5 per high-power field), adequate (5–20 per high-power field), or abundant (>20 per high-power field). Normal platelet numbers in the horse are about half those of other species. In a sample more than a few hours old, platelets may clump into rafts, leaving areas of the slide apparently devoid of platelets. Slides should be scanned for rafts before reporting platelet numbers as low. Enlarged or macroplatelets should also be noted.